Papilloma virus (PV) infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cattle. Papilloma viruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. Papilloma viruses are species specific infective agents; e.g., a human papillomavirus generally does not infect a nonhuman animal.
Papilloma viruses may be classified into distinct groups based on the host that they infect. Human papilloma viruses (HPV) are further classified into more than 60 types based on DNA sequence homology (for a review, see Papilloma Viruses and Human Cancer, H. Pfister (ed.), CRC Press, Inc., 1990). Papilloma virus infections appear to induce type-specific immunogenic responses in that a neutralizing immunity to infection to one type of papilloma virus may not confer immunity against another type of papilloma virus.
In humans, different HPV types cause distinct diseases. HPV types 1,2, 3, 4, 7, 10 and 26-29 cause benign warts in both normal and immunocompromised individuals. HPV types 5, 9, 9, 12, 14, 15, 17, 19-25, 36 and 46-50 cause flat lesions in immunocompromised individuals. HPV types 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata of the genital tract. HPV types 16 and 18 cause epithelial dysplasia of the genital tract and are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal.
Immunological studies in animal systems have shown that the production of neutralizing antibodies to papilloma virus antigens prevents infection with the homologous virus. The development of effective human papilloma virus vaccines has been slowed by the inability to cultivate papilloma viruses in vitro. The development of an effective HPV vaccine has been particularly slowed by the absence of a suitable animal host for the direct study of HPV.
Neutralization of papilloma virus by antibodies appears to be type-specific and dependent upon conformational epitopes on the surface of the virus.
Papilloma viruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for early and late genes. The open reading frames (ORFs) of the virus genomes are designated E1 to E7 and L1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 encode virus capsid proteins. E1 to E3 and E5 to E7 are associated with functions such as viral replication and transformation.
The L1 protein is the major capsid protein and has a molecular weight of 55-60K. L2 protein is a minor capsid protein which has a predicted molecular weight of approximately 55K and an apparent molecular weight of 75-100K as determined by polyacrylamide gel electrophoresis. Electron microscopic and immunologic data suggest that most of the L2 protein is internal to the L1 protein. The L2 proteins are highly conserved among different papilloma viruses, especially the 10 basic amino acids at the C-terminus. The L1 ORF is highly conserved among different papilloma viruses.
The L1 and L2 genes have been used to generate recombinant proteins for potential use in the prevention and treatment of papilloma virus infections. Zhou et al. cloned HPV type 16 L1 and L2 genes into a vaccinia virus vector and infected CV-1 mammalian cells with the recombinant vector to produce virus-like particles (VLP). These studies were interpreted as establishing that the expression of both HPV type 16 L1 and L2 proteins in epithelial cells is necessary and sufficient to allow assembly of VLP. The expression of L1 protein alone or L2 protein alone or double infection of cells with single recombinant vaccinia virus vectors containing L1 and L2 genes did not produce particles.
Bacterially-derived recombinant bovine papilloma virus L1 and L2 have been generated. Neutralizing sera to the recombinant bacterial proteins cross-reacted with native virus at low levels, presumably due to differences in the conformations of the native and bacterially-derived proteins.
Recombinant baculoviruses expressing HPV16 L1 or HPV16 L2 ORF have been used to infect insect SF9 cells and produce recombinant L1 and L2 proteins. Western blot analyses showed that the baculovirus-derived L1 and L2 proteins reacted with antibody to HPV16. The production of HPV 16 L1 and HPV16 L2 proteins by recombinant strains of Saccharomyces cerevisiae has also been demonstrated.
Since cytotoxic T-lymphocytes (CTLs) in both mice and humans are capable of recognizing epitopes derived from conserved internal viral proteins and are thought to be important in the immune response against viruses, efforts have been directed towards the development of CTL vaccines capable of providing heterologous protection against different viral strains.
It is known that CD8.sup.+ CTLs kill virally-infected cells when their T cell receptors recognize viral peptides associated with MHC class I molecules. These peptides are derived from endogenously synthesized viral proteins, regardless of the protein's location or function within the virus. Thus, by recognition of epitopes from conserved viral proteins, CTLs may provide cross-strain protection. Peptides capable of associating with MHC class I for CTL recognition originate from proteins that are present in or pass through the cytoplasm or endoplasmic reticulum. Therefore, in general, exogenous proteins, which enter the endosomal processing pathway (as in the case of antigens presented by MHC class II molecules), are not effective at generating CD8.sup.+ CTL responses.
Efforts to generate CTL responses have included the use of replicating vectors to produce the protein antigen within the cell or have focused upon the introduction of peptides into the cytosol. Both of these approaches have limitations that may reduce their utility as vaccines. Retroviral vectors have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while maintaining the ability of the recombinant virus to replicate, and the effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against the vectors themselves. Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans. Furthermore, the selection of peptide epitopes to be presented is dependent upon the structure of an individual's MHC antigens and, therefore, peptide vaccines may have limited effectiveness due to the diversity of MHC haplotypes in outbred populations.
Intramuscular inoculation of polynucleotide constructs. i.e., DNA plasmids encoding proteins, have been shown to result in the in situ generation of the protein in muscle cells. By using cDNA plasmids that encode viral proteins, antibody responses that provide homologous protection against subsequent challenge can be generated. The use of polynucleotide vaccines (PNVs) to generate antibodies may result in an increased duration of the antibody responses as well as the provision of an antigen that may have the proper post-translational modifications and conformation of the native protein (vs. a recombinant protein). The viral proteins produced in vivo after PNV immunization may assume their native conformation, thereby eliciting the production of virus neutralizing antibody. The generation of CTL responses by this means offers the benefits of cross-strain protection without the use of a live potentially pathogenic vector or attenuated virus.
Benvenisty et al. reported that CaCl.sub.2 precipitated DNA introduced into mice intraperitoneally, intravenously or intramuscularly could be expressed. More recently, intramuscular (i.m.) injection of DNA expression vectors in mice was reported to result in the uptake of DNA by the muscle cells and expression of the protein encoded by the DNA (J. A. Wolff et al., 1990; G. Ascadi et al., 1991). The injected plasmids were shown to be maintained extrachromosomally and did not replicate. Subsequently, persistent expression after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats has been reported. The technique of using nucleic acids as immunogenic agents was reported in WO90/11092 (4 Oct. 1990), in which naked polynucleotides were used to vaccinate vertebrates.
The method is not limited to intramuscular injection. For example, the introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. A jet injector has been used to transfect skin, muscle, fat, and mammary tissues of living animals. Various methods for introducing nucleic acids were reviewed by Donnelly, Ulmer and Liu (The Immunologist, 2:20, 1994).
This invention contemplates a variety of methods for introducing nucleic acids into living tissue to induce expression of proteins. This invention provides methods for introducing viral proteins into the antigen processing pathway to generate virus-specific CTLs and antibodies. Thus, the need for specific therapeutic agents capable of eliciting desired prophylactic immune responses against viral pathogens is met for papilloma virus by this invention. Therefore, this invention provides DNA constructs encoding viral proteins of the human papilloma virus which encode induce specific CTLs and antibodies.
The protective efficacy of DNA vaccination against subsequent viral challenge is demonstrated by immunization with non-replicating plasmid DNA encoding one or more of the above mentioned viral proteins. This is advantageous since no infectious agent is involved, no assembly of virus particles is required, and determinant selection is permitted. Furthermore, because the sequence of some of the gene products is conserved among various types of papilloma viruses, protection against subsequent challenge by a different type of papilloma virus that is homologous to or heterologous to the strain from which the cloned gene is obtained is enabled.